G蛋白信号调控蛋白12在心肌肥厚中的作用及机制研究

发布时间：2018-04-22 04:04

本文选题：心肌肥厚 + 心力衰竭　；参考：《东南大学》2017年博士论文

【摘要】：目的:心肌肥厚是一个多分子、多信号通路参与的复杂病理生理过程,可能导致心力衰竭、猝死等严重心血管事件,但是目前对于心肌肥厚发生发展的分子机制的认识仍十分有限。G蛋白信号通路调节蛋白(RGS)家族多个成员被发现参与心肌肥厚的病理生理过程。RGS12是分子量最大、功能结构域最多的RGS家族成员。结构的复杂性决定了 RGS12可以发挥更复杂和更精密的分子调控作用。本研究旨在阐明RGS12对心肌肥厚的影响及其具体机制。方法:第一部分:选取野生型(WT)小鼠及Sprague-Dawley大鼠乳鼠心肌细胞作为研究对象,分别采用胸主动脉缩窄术(AB)和血管紧张素Ⅱ (Ang Ⅱ)处理建立在体心肌肥厚和离体心肌细胞肥大模型,运用Western blot检测造模后各时间点心肌组织、心肌细胞和成纤维细胞内RGS12表达水平。第二部分:选取全系统RGS12基因敲除(RGS12-KO)小鼠和WT小鼠作为研究对象,采用AB构建心肌肥厚模型。手术4周后检测相关指标:取材小鼠心脏、肺脏和左侧胫骨,计算心体比(HW/BW)、心胫比(HW/TL)和肺体比(LW/BW);采用HE、WGA、PSR等病理染色评价心肌细胞横截面积及胶原沉积水平;超声成像仪检测小鼠心脏功能;应用实时定量PCR检测心肌肥厚和纤维化标志物水平。第三部分:选取心脏特异性RGS12转基因(RGS12-CTG)小鼠和CAG-(loxP)CAT(loxP)-RGSl2/α-MHC-MerCreMer 双转基因(CRMC)小鼠作为研究对象,采用AB构建心肌肥厚模型。手术4周后检测相关指标:取材小鼠心脏、肺脏和左侧胫骨,计算HW/BW、HW/TL和LW/BW;采用HE、WGA、PSR等病理染色评价心肌细胞横截面积及胶原沉积水平;超声成像仪检测小鼠心脏功能;应用实时定量PCR检测心肌肥厚和纤维化标志物水平。第四部分:利用腺病毒转染手段,构建RGS12低表达(AdshRGS12)或高表达(AdRGS12)乳鼠心肌细胞,以腺病毒载体AdshRNA和AdGFP转染乳鼠心肌细胞作为相应的对照组,分别运用Ang Ⅱ和内皮素(ET-1)构建离体心肌细胞肥大模型。促肥厚因子干预48小时后检测相关指标:采用免疫荧光染色比较测量心肌细胞表面积;应用实时定量PCR检测心肌肥厚标志物水平。第五部分:分别在动物实验和细胞实验中,运用Western blot检测各组小鼠心脏组织或心肌细胞中MAPK信号通路分子的总量和磷酸化水平变化。然后,以Sprague-Dawley大鼠乳鼠心肌细胞为研究对象,采用Ang Ⅱ干预构建离体心肌细胞肥大模型,运用免疫共沉淀检测RGS12与信号通路关键分子的相互作用。最后,以RGS12-TG和CRMC小鼠作为研究对象,采用AB构建心肌肥厚模型,使用信号通路分子特异性抑制干预小鼠。手术4周后检测相关指标:取材小鼠心脏、肺脏和左侧胫骨,计算HW/BW、HW/TL和LW/BW;采用HE、WGA、PSR等病理染色评价心肌细胞横截面积及心肌纤维化程度;超声成像仪检测小鼠心脏功能。结果:第一部分:在体心肌肥厚模型和离体心肌细胞肥大模型中,心肌肥厚标志物表达水平明显升高。与假手术组(Sham)相比,AB组小鼠心肌组织内RGS12蛋白表达量明显提高,且手术8周后的表达水平高于手术4周后。与在体实验类似,Ang Ⅱ干预24小时和48小时后,心肌细胞内RGS12的蛋白质水平逐渐升高。然而,Ang Ⅱ干预后,成纤维细胞内RGS12蛋白水平无明显变化。第二部分:WT和RGS12-KO小鼠行假手术造模后,两组小鼠的心脏大小、纤维化程度、心脏功能及相关基因表达水平无明显差异。AB构建心肌肥厚模型4周后,取材结果发现,RGS12-KO小鼠的HW/BW、HW/TL和LW/BW小于WT小鼠;病理染色结果发现,RGS12-KO小鼠的心脏大小、心肌细胞横截面积、间质和血管周围胶原沉积以及胶原容积低于WT小鼠;心脏超声结果发现,RGS12-KO小鼠的左心腔扩大和左心收缩功能减低程度好于WT小鼠;RT-PCR检测结果发现,RGS12-KO小鼠的心肌肥厚标志物的mRNA表达水平低于WT小鼠。第三部分:CRMC和RGS12-CTG小鼠行假手术造模后,两组小鼠的心脏大小、纤维化程度、心脏功能及相关基因表达水平无明显差异。AB构建心肌肥厚模型4周后,取材结果发现,RGS12-CTG小鼠的HW/BW、HW/TL和LW/BW高于CRMC小鼠;病理染色结果发现,RGS12-CTG小鼠的心脏大小、心肌细胞横截面积、间质和血管周围胶原沉积以及胶原容积大于CRMC小鼠;心脏超声结果发现,RGS12-CTG小鼠的左心腔扩大和左心收缩功能减低程度较CRMC小鼠加重;RT-PCR检测结果发现,RGS12-CTG小鼠的心肌肥厚标志物的mRNA表达水平高于CRMC小鼠。第四部分:PBS 干预 AdshRGS12、AdshRNA、AdRGS12 和 AdGFP 组乳鼠心肌细胞48小时后,各组乳鼠心肌细胞表面积和相关基因表达水平无明显差异。Ang Ⅱ干预48小时后,免疫荧光染色结果发现,AdshRGS12组心肌细胞表面积小于AdshRNA组,而AdRGS12组心肌细胞表面积大于AdGFP组;RT-PCR检测结果发现,AdshRGS12组心肌肥厚标志物的mRNA表达水平低于AdshRNA组,而AdRGS12组心肌肥厚标志物mRNA的表达水平高于AdGFP组。第五部分:促肥厚刺激不影响丝裂原活化蛋白激酶(MAPK)信号通路分子的蛋白质总量,却引起MAPK信号通路分子的磷酸化程度升高。其中,JNK1/2和P38的磷酸化程度不受RGS12表达水平的影响,而RGS12高表达提高MEK1/2和ERK1/2的磷酸化水平,RGS12低表达降低MEK1/2和ERK1/2的磷酸化水平。同时,Ang Ⅱ干预后,GS12能够在心肌细胞内与MEK1/2结合形成功能复合体。在RGS-CTG和CRMC小鼠心肌肥厚模型中,MEK1/2特异性抑制剂(U0126)干预能够显著降低小鼠的HW/BW、HW/TL和LW/BW,改善小鼠心肌肥厚、心肌细胞横截面积增大、间质和血管周围胶原沉积,缓解左心腔扩大和左心收缩功能减低。更为重要的是,这些心脏肥厚性重构指标在RGS-CTG/U0126和CRMC/U0126小鼠之间无明显统计学差异。结论:本研究结果表明促肥厚刺激诱导心肌细胞内RGS12表达升高,高表达的RGS12与MEK1/2形成功能复合体,促进MEK1/2-ERK1/2信号通路激活,促进压力负荷诱导的心肌肥厚发生发展。因此,RGS12具有成为临床治疗心肌肥厚新靶点的潜力。
[Abstract]:Objective: myocardial hypertrophy is a complex pathophysiological process involving multiple molecules and multiple signal pathways, which may lead to severe cardiovascular events such as heart failure and sudden death. However, the understanding of the molecular mechanism of the development of cardiac hypertrophy is still limited, and many members of the.G protein signaling family (RGS) family have been found to be involved. The pathophysiological process of muscle hypertrophy (.RGS12) is the largest molecular weight and most functional domain of RGS family. The complexity of the structure determines that RGS12 can play a more complex and more sophisticated molecular regulation. This study aims to elucidate the effect of RGS12 on myocardial hypertrophy and its specific mechanisms. The cardiac myocytes of Sprague-Dawley rats were used as the research object. The models of cardiac hypertrophy and isolated myocardial hypertrophy were established by thoracic aorta coarctation (AB) and angiotensin II (Ang II) respectively. Western blot was used to detect the myocardium, myocardial cells and RGS12 expression of water in the fibroblasts. Second: the second part: select the whole system RGS12 gene knockout (RGS12-KO) mice and WT mice as the research object, construct the myocardial hypertrophy model with AB. After 4 weeks, the related indexes were measured: the heart, the lung and the left tibia were measured, the heart body ratio (HW/BW), the ratio of the tibia (HW/TL) and the lung body ratio (LW/BW) were calculated, and the pathological staining was evaluated by HE, WGA, PSR, etc. The transversal area of cardiac myocytes and the level of collagen deposition; the echocardiography was used to detect the cardiac function of mice; the level of myocardial hypertrophy and fibrosis markers was detected by real-time quantitative PCR. The third part: selected cardiac specific RGS12 transgenic (RGS12-CTG) mice and CAG- (loxP) CAT (loxP) -RGSl2/ a -MHC-MerCreMer double transgenic (CRMC) mice as research The model of cardiac hypertrophy was constructed with AB. After 4 weeks of operation, the related indexes were measured: HW/BW, HW/TL and LW/BW were calculated from the heart, lung and left tibia of mice. The myocardial cell cross section and collagen deposition were evaluated by HE, WGA, PSR and other pathological staining. The cardiac function of mice was detected by the ultrasonic imaging instrument, and the real-time quantitative PCR was used to detect the myocardium. The level of markers of hypertrophy and fibrosis. The fourth part: using adenovirus transfection to construct RGS12 low expression (AdshRGS12) or high expression (AdRGS12) rat myocardial cells, transfection of mammary rat cardiomyocytes with adenovirus vector AdshRNA and AdGFP as the corresponding control group, and use Ang II and endothelin (ET-1) to construct the large model of the isolated cardiomyocytes. Type. After 48 hours of hypertrophy intervention, the related indexes were measured: the surface area of cardiac myocytes was measured by immunofluorescence staining, and the level of myocardial hypertrophy markers was detected by real-time quantitative PCR. The fifth part: in animal experiment and cell experiment, the MAPK signal in heart tissues or cardiac myocytes of each group was detected by Western blot, respectively. The total amount of pathway molecules and the changes in the level of phosphorylation. Then, the rat cardiac myocytes of Sprague-Dawley rats were taken as the research object. Ang II intervention was used to construct the hypertrophy model of isolated cardiomyocytes, and the interaction between RGS12 and the key molecules of the signal pathway was detected by immunoprecipitation. Finally, the RGS12-TG and CRMC mice were used as the research object and AB was used. The model of myocardial hypertrophy was constructed, and the mice were treated with signal pathway molecular specific inhibition. After 4 weeks of operation, the related indexes were measured. HW/BW, HW/TL and LW/BW were calculated from the heart, lung and left tibia of mice. HE, WGA, PSR and other pathological staining were used to evaluate the cross sectional area of myocardial cells and the degree of myocardial fibrosis; the cardiac work of mice was detected by the ultrasonic imaging instrument. Results: the first part: the expression level of the markers of cardiac hypertrophy was significantly increased in the model of body hypertrophy and the hypertrophy of isolated cardiomyocytes. Compared with the sham operation group (Sham), the expression of RGS12 protein in the myocardium of the AB group was significantly increased, and the level of the expression was higher than that after the operation for 8 weeks after 4 weeks. It was similar to that in the body experiment, Ang II. After 24 hours and 48 hours of intervention, the protein level of RGS12 in myocardial cells increased gradually. However, there was no obvious change in the level of RGS12 protein in the fibroblasts after Ang II. Second: after the model of WT and RGS12-KO mice, the size of heart, the degree of fibroid, cardiac function and related gene expression level were not clear in the two groups of mice. 4 weeks after the significant difference of.AB, the results showed that HW/BW, HW/TL and LW/BW in RGS12-KO mice were less than WT mice. Pathological staining showed that the heart size, cross sectional area of cardiac myocytes, interstitial and perivascular collagen deposition and collagen volume of RGS12-KO mice were lower than that of WT mice; cardiac ultrasound findings found RGS12-KO mice The extent of left ventricular enlargement and left cardiac contractile function decreased better than that of WT mice. The results of RT-PCR detection showed that the mRNA expression level of the markers of myocardial hypertrophy in RGS12-KO mice was lower than that of WT mice. The third part: the size, degree of fibrosis, cardiac function and related gene expression in the two groups of mice after the model of CRMC and RGS12-CTG in the model of sham operation. 4 weeks after.AB construction of the myocardial hypertrophy model, the results showed that HW/BW, HW/TL and LW/BW in RGS12-CTG mice were higher than those of CRMC mice. Pathological staining showed that the heart size, cross section area of cardiac myocytes, interstitial and perivascular collagen deposition and collagen volume in RGS12-CTG mice were greater than that of CRMC mice; the echocardiographic results were more than that of CRMC mice. The left heart cavity enlargement and left cardiac contractile function of RGS12-CTG mice were worse than that of CRMC mice. The results of RT-PCR detection showed that the mRNA expression level of myocardial hypertrophy markers in RGS12-CTG mice was higher than that of CRMC mice. The fourth part: PBS intervention AdshRGS12, AdshRNA, AdRGS12 and AdGFP group of milk rat heart cells after 48 hours, each group of milk rat hearts There was no significant difference between the surface area of muscle cells and the expression level of related genes. After 48 hours of.Ang II intervention, the results of immunofluorescence staining found that the surface area of myocardial cells in group AdshRGS12 was less than that of group AdshRNA, and the surface area of cardiac myocytes in group AdRGS12 was larger than that in group AdGFP, and the results of RT-PCR detection found that the mRNA expression level of myocardial hypertrophy markers in group AdshRGS12 was lower than that of group AdGFP. The expression level of mRNA, a marker of myocardial hypertrophy in group AdRGS12, is higher than that in group AdGFP. Fifth: the fifth part: hypertrophy stimulation does not affect the total protein of the mitogen activated protein kinase (MAPK) signaling pathway molecules, but increases the phosphorylation of MAPK signaling molecules. In this, the degree of phosphorylation of JNK1/2 and P38 is not affected by RGS12 expression. The high expression of RGS12 increases the phosphorylation level of MEK1/2 and ERK1/2, and the low expression of RGS12 reduces the phosphorylation level of MEK1/2 and ERK1/2. At the same time, GS12 can combine with MEK1/2 in cardiac myocytes to form functional complexes. In RGS-CTG and CRMC murine cardiac hypertrophy models, MEK1/2 specific inhibitors can intervene. Significantly reduced HW/BW, HW/TL and LW/BW in mice, improved myocardial hypertrophy in mice, increased cross sectional area of cardiac myocytes, interstitial and perivascular collagen deposition, alleviated left ventricular enlargement and left cardiac contractile dysfunction. More importantly, these cardiac hypertrophy indices were not statistically poor between RGS-CTG/U0126 and CRMC/U0126 mice. Conclusion: the results of this study showed that hypertrophy induced the increase of RGS12 expression in cardiac myocytes, high expression of RGS12 and MEK1/2 to form functional complexes, promote the activation of MEK1/2-ERK1/2 signaling pathway and promote the development of myocardial hypertrophy induced by pressure load. Therefore, RGS12 has the potential to be a new target for clinical treatment of myocardial hypertrophy.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.2

【相似文献】